| CPC C12N 5/0656 (2013.01) [C12N 2501/15 (2013.01); C12N 2501/727 (2013.01); C12N 2503/02 (2013.01); C12N 2513/00 (2013.01); C12N 2527/00 (2013.01); C12N 2533/76 (2013.01)] | 19 Claims |
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1. An in vitro quantitative method for proportionally quantifying changes in a 3D extracellular matrix (ECM) mechanics, alignment and organization due to a presence, an amount, a time of contact, a concentration or an absence of a test condition during a synthesis of the ECM, the method comprising the steps of:
(1) obtaining a non-adhesive cell culture well including a non-adhesive peg disposed in the center of the well configured to develop a cellular alignment around the peg during a culture of anchorage-dependent cells and self-assembling cells, whereby the anchorage-dependent and self-assembling cells are operative to grow around the peg and self-aggregate and self-assemble using only one or more cell-derived forces;
(2) seeding the culture well with a culture medium and the anchorage-dependent cells and the self-assembling cells, wherein the cells are configured to synthesize ECM components and the ECM during a cultivation;
(3) cultivating the cells, whereby the cells grow and adhere to one another without an application of an external force, an anchorage force/material and a scaffolding material, but the cells do not adhere to the well and do not adhere to the peg; whereby the cells exert cytoskeleton-mediated tension and cell-driven self-assembly to form a 3D ring-shaped geometry of tissue ECM around the peg; wherein the ring-shaped geometry of ECM includes the tension, the cell-driven self-assembly, the cellular alignment around the peg from steps (1)-(3), and mechanics and organization that are all free of an effect from an external force, anchorage force/material and a scaffolding material thereby providing the ECM with a no cell-to-scaffold biophysical crosstalk and a no cell-to-external force biophysical crosstalk;
(4) measuring one or more properties of the tissue ECM to obtain a proportional signal for each of the one or more properties;
(5) providing a test condition and repeating steps (1)-(4) with a contacting of the cells with the test condition by contacting the tissue ECM and/or cells with an agent, a force, or a factor; whereby the presence, amount, time of contact, concentration or absence of the test condition either does or does not cause a proportional and quantitative, un-predicted, observable and measurable change in the one or more properties of the ECM and whereby the measuring in step (4) measures the proportional signal that is a quantitative response to the presence, amount, time of contact, concentration or absence that causes a proportional change in the one or more properties, then comparing the proportional signal before the contacting and the proportional signal after the contacting;
wherein the method is configured to provide tissue ECM that does not cause a noise value and/or a crosstalk value in the signal of the measuring of the change from the test condition because of an absence of a scaffolding material, and anchorage force/material and an externally applied force during the execution of steps (1), (2), (3), (4) and the method is configured to provide an absence of a noise value including a no cell-to-scaffold biophysical crosstalk signal and a no cell-to-external force biophysical crosstalk signal; and wherein the absence of the noise value and/or the crosstalk values provides and enables a quantitative method that is proportionally responsive to a presence of test condition, an absence of test condition, a time of contact of test condition applied and/or a difference in amount/magnitude of the test condition.
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